Definición de iniciativas e indicadores propuestos

At least 30 genes (ATGs) were identified in yeast that function in the regulation and execution of autophagy (Xie et al., 2007). Some of these genes are known homologous in mammalian cells. Classical autophagy can be subdivided into four main phases that are performed by specific ATG genes (Levine et al.; 2008; Xie et al.; 2007). Among the autophagy-related (ATG) genes, discovered in yeast and almost integrally conserved in all eukaryotic phyla, which control the formation of the autophagosome (Klionsky et al.; 2003), beclin 1 (the mammalian ortholog of the yeast ATG6) is a tumor suppressor gene that contributes with the class III phosphatidylinositol-3- kinase (PI3K) to the formation of the autophagosome (Liang et al.; 1999; Kihara et al.; 2001). Other tumor suppressor gene products, such as p53, PTEN, TSC1/TSC2, death- associated protein kinase (DAP kinase), are involved in the control of autophagy (Botti et al.; 2006). Interestingly, autophagy is also stimulated in cancer cells by ceramide (Scarlatti et al.; 2004; Daido et al.; 2004), a tumor suppressor lipid (Hannun et al.; 1993).

Although autophagy is a dynamic process, the pathway was delineated into several static steps for the convenience of description: induction and nucleation, phagophore expansion, autophagosome targeting, docking and fusion, and cargo degradation and recycling.

Many signaling pathways and proteins seem to be involved in this first phase, but the esact mechanism of action and/or interaction is unknown. Autophagy may be induced as a response to a change in the extracellular environment of a cell, and the target of rapamycin complex 1 (TORC1) is one of the signaling pathways that plays a primary role in sensing the shift

in nutrient availability. Nutrient starvation, in particular nitrogen and/or amino acid limitation, initiates an intracellular signalling cascade by discontinuing TORC1 stimulation, resulting in the activation of the Atg1 kinase complex. The Atg1 kinase complex works directly downstream of the TORC1 pathway and it consists of Atg1, Atg13, and a scaffold subcomplex that includes Atg17- Atg31-Atg29 (Kamada et al.; 2000). Assembly of this complex is crucial for autophagy because it plays a role in recruiting other Atg proteins to the phagophore assembly site (PAS) and activating downstream targets through phosphorylation (Suzuki et al.; 2007; Papinski et al.; 2014). Protein kinase A (PKA) is another negative regulator of the Atg1 kinase complex, in this case primarily in response to carbon source, whereas the energy sensor Snf1/AMP-activated protein kinase (AMPK) acts as a positive regulator. Although the associations among these proteins have been demonstrated as biomolecular interactions, it is unknown whether all of these proteins are ever present in a single complex. In autophagy, nucleation refers to the process of mobilizing a small group of molecules to the PAS; the phagophore is the active sequestering compartment of autophagy. The class III PtdIns3K complex I, which is employed specifically for autophagy, is one of the key complexes that are recruited to the PAS upon induction of autophagy. This complex is constituted of five distinct proteins: the lipid kinase Vps34, the regulatory kinase Vps15, Vps30/Atg6, Atg14 and Atg38, which are all necessary for autophagy (Schu et al.; 1993; Kihara et al.; 2001). In brief, the class III PtdIns3K is responsible for the production of phosphatidylinositol-3-phosphate (PtdIns3P) directly from phosphatidylinositol (Burman et al.; 2010). This PtdIns3P is important for

the correct localization of some of the Atg proteins including Atg18 and Atg2, which enables the recruitment of Atg8, Atg9 and Atg12 to the PAS (Obara et al.; 2008). Other proteins and molecules that play a role in regulating this first autophagic phase are p53, c-jun-N-terminal kinase1 (JNK1), eucariotic initiation factor 2α (elF2α), GTPase and intracellular calcium (Levine et al 2008; Talloczy et al.; 2009;). All these molecules and/or signaling pathways appear to promote autophagy by activating class III PtdIns3K Cps3, which promotes the formation of phosphatidylnositous 3-phosphate (PIP3) on lipids. Autophagosomes, which correspond to the mature form of the phagophore, is essentially a terminal compartment that does little more than fuse with the vacuole; formation of the phagophore and sequestration by the phagophore are the truly dynamic steps of autophagy (Baba et al.; 1994). There are two essential ubiquitin-like (Ubl) conjugation systems that are necessary for phagophore expansion and these involve the Ubl proteins Atg12 and Atg8 (Ohsumi; 2001); these two proteins have structural similarity to ubiquitin, but are not actual homologs. Atg12 is conjugated to Atg5 via the action of the E1 and E2 enzymes Atg7 and Atg10, and this conjugate binds Atg16 to form the dimeric Atg12–Atg5–Atg16 complex; Atg8 undergoes a different type of conjugation, being covalently attached to the lipid phosphatidyl- ethanolamine (PE). The generation of Atg8–PE involves the protease Atg4, Atg7 as an E1 enzyme and Atg3 as an E2 enzyme, with the Atg12– Atg5–Atg16 complex participating as an E3 enzyme, although the latter is not absolutely required for conjugation to occur (Cao et al.; 2008). A detailed mechanism in which these conjugation systems operate along with other complexes to enlarge the phagophore is currently an on-going

research topic. Atg9 functions in some manner as the membrane transporter for the growing phagophore, but direct evidence or a mechanistic explanation are not available. Nonetheless, Atg9 has multiple functions; first, Atg9 is the only transmembrane protein that is essential for phagophore expansion (Noda et al.; 2000); second, Atg9 is found to be highly mobile in the cytosol upon rapamycin treatment (Yamamoto et al.; 2012); third, this protein is capable of binding with itself and appears to transit to the PAS as part of a complex (Reggiori et al.; 2005). While none of these studies directly proves the role of Atg9 in membrane shuttling, researchers have begun identifying the machinery that is involved in Atg9 trafficking. Upon completion of the autophagosome, it fuses with the vacuole. This fusion allows the release of the inner autophagosome vesicle into the vacuole lumen where it is now termed an autophagic body. Note that mammalian cell lysosomes are generally smaller than autophagosomes so autophagic bodies are not a general feature of autophagy in most of the more complex eukaryotes. The mechanism that controls the fusion is unknown at present; however, there are regulatory mechanisms to prevent premature autophagosome fusion with the vacuole, which would prevent delivery of the cargo into the vacuole lumen. Other cellular processes that also deliver their cargo to the vacuole employ similar components that facilitate fusion including SNARE (SNAP= Soluble NSF Attachment Protein Receptor) proteins and those involved in the homotypic fusion and vacuole protein sorting (HOPS) pathway (Jiang et al.: 2014). After the cargo is delivered inside the vacuole, the autophagic body membrane is degraded by a putative lipase, Atg15, (Epple et al.; 2001; Teter et al.; 2001) followed by cargo degradation by resident hydrolases. Once

degraded, the resulting macromolecules (aminoacids, proteins and lipids) are released back into the cytosol through various permeases including Atg22 (Yang et al.; 2006).